THP-1 Monocyte Extracellular Vesicles Exhibit Antibiofilm Effects on Pseudomonas aeruginosa

Introduction: Antimicrobial resistance is implicated annually in over 35,000 deaths in the US and 1.27 million deaths worldwide. Thus, the need for new antimicrobial approaches is urgent and clear. A particular pathogen of concern is multidrug resistant Pseudomonas aeruginosa (PA), which is responsible for over 32,000 infections and 2,700+ deaths in the US yearly. One potential class of novel therapeutics is extracellular vesicles (EVs), which have been found to have intrinsic antimicrobial and anti-virulence properties. EVs constitute a platform with inherent therapeutic properties that can act synergistically with direct-acting antimicrobials enabling combined direct and anti-virulence therapeutic delivery. EVs may overcome certain challenges associated with novel antimicrobial therapeutics by: 1) delivering a broad range of multifunctional cargo, thus constraining microbial resistance, and 2) improving the pharmacokinetic profile of antimicrobial cargos. Here, the antimicrobial activity of EVs on PA was explored in the context of biofilms of hypervirulent strains PA14 and PAO1. Biofilms are communities of microbes that grow at interfaces or on surfaces and biofilms comprise approximately 80% of all infections. Preliminary studies identified human monocyte cell line THP-1 as a promising source of EVs for this application. In this study EVs derived from THP-1 monocytes as well as from differentiated THP-1 macrophages in their basal, inflammatory, and regenerative states were tested. I hypothesize that EVs derived from differentiated THP-1s will have varied effect on biofilm formation, with inflammatory macrophage derived EVs reducing biofilm formation the most, as this is the subtype associated with infection fighting.

Materials and

Methods: THP-1 cells were cultured and differentiated into macrophages with 20 nM PMA treatment. Subsequently, inflammatory macrophages were polarized with 10 pg/mL LPS and 20 ng/mL IFNg treatment and regenerative macrophages were differentiated with 20 ng/mL IL4 and IL13. The THP-1 EVs were isolated using differential centrifugation and tangential flow filtration and analyzed using nanoparticle tracking analysis to determine size and concentration. These EVs were incubated with PA cells in a microtiter plate at a dose of 5E9 EVs/mL for 24 hours to allow biofilm formation. Then the biofilm was washed with water and stained with crystal violet. After allowing the stained biofilms to dry overnight, the crystal violet was solubilized in 30% acetic acid. The absorbance was quantification with spectrophotometry to assess biofilm formation in the presence of THP-1 EVs. The quantified absorbance measurements were compared with a one-way ANOVA with alpha equal to 0.05 using Prism GraphPad.

Results, Conclusions, and Discussions: THP-1 EVs were shown to reduce PA14 biofilm formation compared to negative controls. The negative controls included a vehicle, no treatment (PBS) control and RPMI media control. The RPMI media control was utilized to account for the effects of co-isolated media contaminants. The control is volume matched media that undergoes the EV isolation process without touching cells. The anti-biofilm effect for the THP-1 EVs was shown to be dose dependent. The antibiofilm effect was present for all THP-1 and differentiated THP-1 EV treatments with no statistical differences found between the treatment groups. THP-1 EVs decreased biofilm formation by 25.6%, M0 EVs by 20.6%, M1 EVs by 24.7%, and M2 EVs by 27.9%. Additionally, through measurement of the OD600 of the planktonic culture, THP-1 EVs were not shown to decrease PA14 growth, indicating an anti-virulence effect. Overall, these data implicate THP-1 EVs and differentiated THP-1 EVs as potential antibiofilm therapeutics of interest, with mechanistic investigations still ongoing. Current work aims to study whether the THP-1 EV treatments affect the metabolic activity of the PA cells and what components of the EVs are necessary for the anti-biofilm effect. Additionally, I aim to determine what kind of interactions occur between the THP-1 EVs and the PA cells. Traditionally, EVs are thought to be functional by delivering therapeutic cargo to recipient cells. However, there is little understanding of the interactions that can occur between host cell derived EVs and bacterial pathogens. Microbial BEVs have been shown to exact damage on host cells and modulate their response to infections, so it is plausible to assume that the reverse could be true. With my future work, I aim to understand the mechanism of the effect and methods for enhancing it so EVs can be validated as a potential anti-biofilm treatment.

Acknowledgements (Optional):